Loss of Ift74 Leads to Slow Photoreceptor Degeneration and Ciliogenesis Defects in Zebraﬁsh

International Journal of Molecular Sciences

Article Loss of Ift74 Leads to Slow Photoreceptor Degeneration and Ciliogenesis Defects in Zebraﬁsh

Panpan Zhu 1,† , Jingjin Xu 1,†, Yadong Wang 1 and Chengtian Zhao 1,2,3,*

1 Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; [email protected] (P.Z.); [email protected] (J.X.); [email protected] (Y.W.) 2 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266003, China 3 Sars-Fang Centre, Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China * Correspondence: [email protected] † These authors have contributed equally to this work.

Abstract: Cilia are microtubule-based structures projecting from the cell surface that perform diverse biological functions. Ciliary defects can cause a wide range of genetic disorders known collectively as ciliopathies. Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia by transporting proteins along the axoneme. Here, we report a lack of Ift74, a core IFT-B protein, leading to ciliogenesis defects in multiple organs during early zebrafish development. Unlike rapid photoreceptor cell death in other ift-b mutants, the photoreceptors of ift74 mutants exhibited a slow degeneration process. Further experiments demonstrated that the connecting cilia of ift74 mutants were initially formed but failed to maintain, which resulted in slow opsin transport efficiency and eventually led to photoreceptor cell death. We also showed that the large amount of maternal ift74 transcripts deposited in zebrafish eggs account for the main reason of slow photoreceptor Citation: Zhu, P.; Xu, J.; Wang, Y.; degeneration in the mutants. Together, our data suggested Ift74 is critical for ciliogenesis and that Zhao, C. Loss of Ift74 Leads to Slow Ift proteins play variable roles in different types of cilia during early zebrafish development. To our Photoreceptor Degeneration and Ciliogenesis Defects in Zebrafish. Int. knowledge, this is the first study to show ift-b mutant that displays slow photoreceptor degeneration J. Mol. Sci. 2021, 22, 9329. https:// in zebrafish. doi.org/10.3390/ijms22179329 Keywords: zebrafish; ift74; cilia; photoreceptor degeneration; opsin transport Academic Editors: Seong-Kyu Choe and Cheol-Hee Kim

Received: 24 July 2021 1. Introduction Accepted: 23 August 2021 Retinal degeneration is one of the major causes of human blindness due to progressive Published: 28 August 2021 death and dysfunction of photoreceptors. The vertebrate photoreceptor cell is a specialized type of neuroepithelial cell with a distinctive morphology, consisting of an outer segment Publisher’s Note: MDPI stays neutral (OS), inner segment (IS), the nuclear region, and synapse. OS can be recognized as a highly with regard to jurisdictional claims in modified sensory cilium that is rich in the photosensitive G protein-coupled receptors published maps and institutional affil- (GPCRs), opsins. The OS and IS are connected through a narrow structure called the iations. connecting cilium, through which opsin and other OS protein components synthesized in the IS can be transported into the OS [1,2]. Cilia are highly conserved organelles extending from the surface of almost all vertebrate cells. Generally, cilia are often classified as either motile or primary (sensory) cilia. Copyright: © 2021 by the authors. Motile cilia in the respiratory tract help the transport of mucus toward the throat, and Licensee MDPI, Basel, Switzerland. movement of ependymal cilia creates a flow of cerebrospinal fluid in the brain ventricles. This article is an open access article The primary cilia function as cellular antenna in that they are enriched for multiple mem- distributed under the terms and brane receptors for sensing extracellular signals, such as growth factors, hormones, odor conditions of the Creative Commons molecules, and developmental morphogens [3–5]. Defects in cilia formation and/or func- Attribution (CC BY) license (https:// tion can lead to a wide range of inherited ciliopathic diseases in humans, including retinal creativecommons.org/licenses/by/ situs inversus 4.0/). degeneration, kidney disease, brain abnormalities, , and other defects [6].

Int. J. Mol. Sci. 2021, 22, 9329. https://doi.org/10.3390/ijms22179329 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9329 2 of 13

The assembly, maintenance, and function of the cilia require bidirectional movement of protein complexes along the microtubule-based axonemes. Ciliogenesis requires intraflagellar transport (IFT) to mediate the delivery of components such as tubulin, dyneins, and membrane proteins to the tip of the cilia, where new cilia assembly occurs [7]. The IFT particle is formed by two complexes, complex A and complex B, consisting of at least 6 and 14 subunits, respectively. In the anterograde direction, the IFT complex binds to kinesin-2 heterotrimer through IFT-B to transport related substances to the tip of cilia, while transport from cilia tip to base (retrograde) is mediated by IFT complex A (IFT-A) and the cytoplasmic dynein motor [7,8]. In vertebrates, the antegrade Kinesin-2 family contains four members: KIF3A, KIF3B, KIF3C, and KIF17. While KIF17 forms homodimers for fast, highly processive transport, KIF3A and KIF3B are normally found in a heterotrimeric complex together with the kinesin-associated protein 3 (KAP3) [9]. In zebrafish and mouse, KIF3 heterotrimer motors play a major role during ciliogenesis as cilia are still present in mutants lacking KIF17 proteins [9–11]. Being a modified cilium, it is easy to speculate that mutations of either IFT components or Kinesin motors can affect the formation of outer segments, thus interrupting the function of photoreceptor cells and leading to visual loss. The conditional knockout of KIF3A in a mouse photoreceptor cell layer results in dysplasia of the connecting cilia, which in turn lead to apoptosis of rods and cones [12,13]. In zebrafish, both kif3a and kif3b mutants display abnormal photoreceptor development at early developmental stages. Interestingly, rod photoreceptors degenerate rapidly while cones undergo a slow degenerate process in these kinesin-2 mutants. An abnormal accumulation of opsins in the rods cell membrane due to insufficient transport is the major cause of fast rods degeneration [11]. IFT plays a critical role during OS assembly and mutations in genes encoding IFT proteins, such as IFT88 and IFT172, causing severe ablation or disruption of OS [14–17]. Of note, the photoreceptor degeneration process is less severe in the mutants of IFT-A components than those of IFT-B components, which is consistent with the concept that components of the IFT-B subunit are essential for ciliogenesis [18]. IFT74, together with its short protein variant, IFT72, forms a heterotetramer with IFT81, composing the backbone of the IFT-B core complex. The interaction between IFT74 and IFT81 proteins forms a module that is essential for the binding of tubulin, the backbone, and the most abundant ciliary proteins [19]. Recently, IFT74 has been identified as causative for human ciliopathies, Bardet–Biedl syndrome (BBS), and Joubert syndrome (JBTS) [20–22]. The role of IFT74 during embryonic development remains to be elucidated. Here, we identified zebrafish ift74 mutants and reported ciliogenesis defects in most organs. Unexpectedly, mutant photoreceptor cells displayed slow onset progressive degeneration as late as 5 days post fertilization, which is different from the rapid photoreceptor degeneration occurring in other IFT-B core complex mutants. We further examined the reasons for slow photoreceptor degeneration in ift74 mutants and uncovered variable requirements of maternal proteins during early ciliogenesis.

2. Results 2.1. Zebraﬁsh michelin Locus Encodes ift74, an IFT-B Component The michelin mutant is a spontaneous line occurring in our lab, and the mutant is characterized by the presence of a ventral body axis (Figure1A,B). We initially named this line as michelin because the bended tail resembles a tire. Approximately 25% of the produced embryos from two intercrossed michelin heterozygotes showed body curvature phenotype (Figure1C), suggesting that these mutants were caused by the mutation of a single allele. Next, we performed linkage analysis and mapped the mutation to a region in linkage group 5 (LG5) deﬁned by microsatellite Z8921 (10 recombination in 24 meiotic events) and Z50336 (0 recombination in 23 meiotic events, Figure1D). We found that ift74, encoding a component of the IFT-B core complex, is located in this region. To assess whether michelin harbors a mutation in the ift74 gene, we performed PCR analysis to amplify the open reading frame (ORF) of the ift74 gene from wild-type, michelin mutant, Int. J. Mol. Sci. 2021, 22, 9329 3 of 14

events) and Z50336 (0 recombination in 23 meiotic events, Figure 1D). We found that ift74, Int. J. Mol. Sci. 2021, 22, 9329 encoding a component of the IFT‐B core complex, is located in this region. To assess3 of 13 whether michelin harbors a mutation in the ift74 gene, we performed PCR analysis to am‐ plify the open reading frame (ORF) of the ift74 gene from wild‐type, michelin mutant, and theirand their sibling sibling embryos. embryos. The The results results showed showed that that no no band band can can be be amplified ampliﬁed from from michelin homozygotes (Figure 11E).E). FurtherFurther PCRPCR resultsresults fromfrom genomicgenomic DNADNA showedshowed thatthat only only the the firstﬁrst exon was still present, while the remaining exons were absent in the mutant genome (Figure S1A). S1A). These These results results imply imply that that there there may may have have a chromosomal a chromosomal rearrangement rearrangement oc‐ curringoccurring in the in the ift74ift74 locuslocus in michelin in michelin mutants,mutants, which which interrupts interrupts the transcription the transcription of the of ift74 the gene.ift74 gene.

Figure 1. michelin encodes a zebrafish zebrafish homolog of the ift74 gene. ( A,B) External phenotypes of wild wild-type‐type (wt) and michelin mutants at 5 dpf. (C) Bar graph showing the percentages of embryos with body curvature defects in different groups as mutants at 5 dpf. (C) Bar graph showing the percentages of embryos with body curvature defects in different groups as indicated. SP, splicing morpholino; ATG, translation‐blocking morpholino. (D) Genetic map on LG 5 showing the number indicated. SP, splicing morpholino; ATG, translation-blocking morpholino. (D) Genetic map on LG 5 showing the number of recombinants near michelin/ift74 locus. The exon/intron structure of the ift74 transcript was shown in the middle. Bottom ofshowing recombinants the sequence near michelin/ift74 of wild‐typelocus. and Theift74 exon/intron mutants generated structure with of the CRISPR/Cas9ift74 transcript methods. was shown The insgRNA the middle. targeting Bottom site showingwas indicated the sequence with overline, of wild-type the PAM and siteift74 wasmutants labeled generated in green and with the CRISPR/Cas9 changing bases methods. in the target The sgRNA region targeting were shown site wasin red. indicated (E) PCR with analysis overline, showing the PAM the amplification site was labeled of ift74 in green ORF andin 3 dpf the changingwild‐type, bases sibling, in the and target michelin region mutant were larvae. shown (F in– red.H) External (E) PCR phenotypes analysis showing of 5 dpf the ift74 amplification mutant and of morphantift74 ORF larvae in 3 dpf as wild-type, indicated. sibling, Scale bars: and 500michelin μm. mutant larvae. (F–H) External phenotypes of 5 dpf ift74 mutant and morphant larvae as indicated. Scale bars: 500 µm. To further prove that the mutation of ift74 can result in michelin mutant phenotypes, we generatedTo further zebrafish prove that ift74 the mutants mutation with of ift74the CRISPR/Cas9can result in michelinmethod.mutant The sgRNA phenotypes, target‐ ingwe generatedthe second zebrafish exon of ift74 mutantswas used with to introduce the CRISPR/Cas9 a double method.‐stranded The break, sgRNA which targeting led to geneticthe second mutations exon of dueift74 to wassubsequently used to introduce non‐homologous a double-stranded end‐joining break, (NHEJ) which repair. led We to recoveredgenetic mutations a mutant due line to carrying subsequently 4 bp deletion non-homologous in the target end-joining region, causing (NHEJ) a repair.frameshift We mutationrecovered of a mutantthe ift74 line gene carrying during 4mRNA bp deletion translation in the (Figure target region, 1D). The causing expression a frameshift of mu‐ tantmutation messenger of the RNAift74 gene was duringalso decreased mRNA translationin the mutants (Figure due1 D).to nonsense The expression mediated of mutant decay, whichmessenger further RNA confirmed was also decreasedthe specificity in the of mutants this mutant due to(Figure nonsense S1B,C). mediated Similar decay, to michelin which mutants,further confirmed mutation the of the specificity ift74 gene of this also mutant led to strong (Figure body S1B,C). curvature Similar todefectsmichelin (Figuremutants, 1F). Inmutation addition, of thecrossingift74 gene between also led michelin to strong and body ift74 curvature heterozygotes defects also (Figure produced1F). In addition,approxi‐ crossing between michelin and ift74 heterozygotes also produced approximately 25% (42 in mately 25% (42 in 184) of embryos with body curvature defects (Figure 1C,G). Finally, 184) of embryos with body curvature defects (Figure1C,G). Finally, knocking down of ift74 with two individual morpholinos to block translation (ATG-MO) and splicing (SP-MO) also caused embryos to develop a ventral curly body axis (Figure1C,H). Taken together, these results provide convincing evidence that michelin encodes ift74. In the following study, we performed all analysis using ift74 mutants. Int. J. Mol. Sci. 2021, 22, 9329 4 of 14

knocking down of ift74 with two individual morpholinos to block translation (ATG‐MO) and splicing (SP‐MO) also caused embryos to develop a ventral curly body axis (Figure 1C,H). Taken together, these results provide convincing evidence that michelin encodes

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2.2. Ciliary Defects in Multiple Organs of ift74 Mutants

2.2.Considering Ciliary Defects that in Ift74 Multiple is one Organs of the of ift74 core Mutants IFT‐B complex required for ciliogenesis, we first analyzed cilia development in ift74 mutants. Cilia are present in diverse organs of Considering that Ift74 is one of the core IFT-B complex required for ciliogenesis, we developingfirst analyzed zebrafish, cilia development including olfactory in ift74 mutants. placode, Cilia pronephric are present duct, in diverse ventral organs spinal of cord, ear,developing photoreceptor, zebrafish, and including neuromasts olfactory (Figure placode, 2A) pronephric[23]. To directly duct, ventral visualize spinal cilia, cord, we ear, per‐ formedphotoreceptor, whole‐mount and neuromasts immunocytochemistry (Figure2A) [ 23 with]. To directlyanti‐glycylated visualize α‐ cilia,tubulin we performed antibody on 3 andwhole-mount 5 days post immunocytochemistry fertilization (dpf) zebrafish with anti-glycylated larvae. Comparedα-tubulin with antibody those in on sibling 3 and em‐ bryos,5 days cilia post displayed fertilization various (dpf) defects zebrafish in larvae.ift74 mutants Compared (Figures with those 2 and in S2). sibling Cilia embryos, in nasal pit andcilia spinal displayed cord were various nearly defects absent in ift74 in mutantsthe mutants, (Figure while2 and cilia Figure in S2).hair Cilia cells in of nasal ear macula, pit ear andcrista, spinal and cord neuromast were nearly are absentstill present in the mutants, but shorter. while In cilia the in pronephric hair cells of duct, ear macula, multicilia ear crista, and neuromast are still present but shorter. In the pronephric duct, multicilia were shorter and disorganized in ift74 mutants (Figures 2 and S2). These results suggest were shorter and disorganized in ift74 mutants (Figure2 and Figure S2). These results thatsuggest Ift74 is that essential Ift74 is for essential the development for the development of cilia ofin ciliaall examined in all examined organs. organs.

FigureFigure 2. Cilia 2. defectsCilia defects in ift74 inmutants.ift74 mutants. (A) Representative (A) Representative image image showing showing the the position position of ofcilia cilia in indifferent different organs organs as as indi‐ cated inindicated a 5 dpf in zebrafish a 5 dpf zebraﬁsh larva. ( larva.B–G′) (B Confocal–G0) Confocal images images showing showing cilia cilia in in different different organs of of wild-type wild‐type and andift74 ift74mutants mutants as indicated.as indicated. Cilia Ciliawere were visualized visualized with with anti anti-glycylated‐glycylated tubulin tubulin antibody antibody (green),(green), and and nuclei nuclei were were counterstained counterstained with with DAPI (blue).DAPI (blue). OP, olfactory OP, olfactory placode; placode; EM, EM, ear ear macula; macula; EC, EC, ear ear crista; crista; NM, NM, neuromast; PD PD pronephric pronephric duct; duct; SC, SC, spinal spinal canal. canal. Scale bars:Scale ( bars:A), 500 (A), μ 500m; µ(Bm;′–G (B′0),– G100), μ 10m.µ m.

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2.3. Slow-Progressing Photoreceptor Cell Degeneration in ift74 Mutant 2.3. Slow‐Progressing Photoreceptor Cell Degeneration in ift74 Mutant Photoreceptor outer segments are highly modiﬁed sensory cilia for phototransduc- Photoreceptor outer segments are highly modified sensory cilia for phototransduc‐ tion. We examined photoreceptor development in ift74 mutants. Immunostaining results tion. We examined photoreceptor development in ift74 mutants. Immunostaining results with zpr-1 antibody, which selectively labels cell bodies of the red- and green-sensitive with zpr‐1 antibody, which selectively labels cell bodies of the red‐ and green‐sensitive double cones [24], showed that the numbers of double cones per retinal cross-section were double cones [24], showed that the numbers of double cones per retinal cross‐section were comparablecomparable between between wild-type wild‐type and and mutant larvae larvae at at 5 dpf 5 dpf (Figure (Figure 3A,B).3A,B). At 7 At dpf 7 and dpf 10 and 10 dpf,dpf, the the number number ofof doubledouble cones decreased decreased significantly signiﬁcantly (Figure (Figure 3A,B).3A,B). The The staining staining was was almostalmost completely completely disappeared disappeared in in the the center center retinaeretinae of 10 10 dpf dpf mutant mutant larvae, larvae, suggesting suggesting thatthat the majoritythe majority of cone of cone photoreceptors photoreceptors degenerated degenerated at at this this time time (Figure (Figure3A,B). 3A,B).Meanwhile, Mean‐ the lengthwhile, ofthe the length cell bodyof the was cell body significantly was significantly shorter in shorter the mutants in the starting mutants at starting 5 dpf (Figure at 5 dpf3 C). We further(Figure 3C). evaluated We further outer evaluated segments outer formation segments in formation the mutants in the by mutants staining by with staining wheat germwith agglutinin wheat germ (WGA) agglutinin or zpr3 (WGA) antibody, or zpr3 which antibody, labels which all the labels outer all segments the outer orsegments rod outer segments,or rod respectively.outer segments, Similarly, respectively. photoreceptor Similarly, photoreceptor outer segments outer were segments initially were formed ini‐ at 5 dpf,tially while formed they at degenerated5 dpf, while they during degenerated development during development in both ift74 inand bothmichelin ift74 andmutants mich‐ (Figureelin mutants S3). These (Figure results S3). suggested These results that suggested photoreceptors that photoreceptors were initially were formed initially and formed endured slowand progressive endured slow degeneration progressive in degeneration the absence in of the Ift74 absence protein. of Ift74 protein.

FigureFigure 3. Slow 3. photoreceptorSlow photoreceptor degeneration degeneration in ift74 in ift74mutants. mutants. (A ()A Confocal) Confocal images images showingshowing the the cell cell bodies bodies of of red red and and green green doubledouble cones cones in wild-type in wild‐type and ift74and ift74mutant mutant larvae larvae at differentat different stages stages as as indicated. indicated. The double cones cones were were stained stained with with zpr-1 antibodyzpr‐1 antibody (green), (green), and nucleiand nuclei were were stained stained with with DAPI DAPI (blue). (blue). Enlarged Enlarged viewsviews of the the boxed boxed area area are are shown shown on on the the right. (B,C) Statistical results showing the number of cones per retina section and length of cone cell body in different B C right. (groups, ) Statistical as indicated. results Measurements showing the of the number cell body of cones length per are retinashown sectionin panel and (A) length(Red square of cone brackets). cell body Scale in bars: different 20 groupsμ asm. indicated. **** p < 0.0001, Measurements ns, no significant. of the cell body length are shown in panel (A) (Red square brackets). Scale bars: 20 µm. **** p < 0.0001, ns, no signiﬁcant. Considering that cilia were defective in multiple organs as early as 3 dpf, the slow photoreceptorConsidering degeneration that cilia were in the defective mutants in was multiple quite surprising, organs as especially early as 3 when dpf, multi the slow‐ photoreceptorple mutant degenerationlines of the IFT in‐B thecomplex mutants have was been quite reported surprising, to exhibit especially severe photoreceptor when multiple mutantdegeneration lines of theat 5 dpf IFT-B [14,16,25]. complex Therefore, have been we compared reported photoreceptor to exhibit severe survival photoreceptor between degenerationift74, ift88, atand 5 kif3a dpf [mutants.14,16,25 ].Ift88 Therefore, is a member we comparedof the IFT‐B photoreceptor complex and Kif3a survival is the betweenmajor ift74, ift88, and kif3a mutants. Ift88 is a member of the IFT-B complex and Kif3a is the major motor protein required for IFT in vertebrate cilia. In contrast to the slow progressive

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degeneration in ift74 mutants, both ift88 and kif3a mutants displayed rapid photoreceptor motor protein required for IFT in vertebrate cilia. In contrast to the slow progressive de‐ degenerationgeneration in at ift74 5 dpf mutants, zebrafish both larvae ift88 and (Figure kif3a S4). mutants displayed rapid photoreceptor degeneration at 5 dpf zebrafish larvae (Figure S4). 2.4. Ift74 Is Required for the Maintenance of Connecting Cilium 2.4.To Ift74 understand Is Required thefor the reasons Maintenance for the of Connecting slow progressive Cilium degeneration of photoreceptor cells inToift74 understandmutants, the we reasons evaluated for the slow formation progressive and maintenance degeneration of photoreceptor con- nectingcells in cilium. ift74 mutants, Immunostaining we evaluated results the formation using anti-acetylated and maintenanceα-tubulin of photoreceptor antibody con showed‐ thatnecting the number cilium. andImmunostaining length of connecting results using cilia anti were‐acetylated grossly α‐ normaltubulin antibody in the retinae showed of 3 dpf mutantthat the larvae number (Figure and length4A–C). of At connecting 5 dpf, the cilia number were grossly of connecting normal in ciliathe retinae began of to 3 decreasedpf significantlymutant larvae and (Figure was gradually 4A–C). At lost 5 dpf, during the number development of connecting (Figure cilia4B). began Notably, to decrease the remain- ingsignificantly cilia in the and mutant was photoreceptorsgradually lost during were development still of similar (Figure length 4B). with Notably, those inthe wild-type re‐ larvaemaining (Figure cilia4 C).in the In mutant contrast, photoreceptors connecting ciliawere were still of absent similar in length both withift88 thoseand kif3ain wildmutants‐ (Figuretype 4larvaeA). These (Figure results 4C). In further contrast, confirmed connecting that cilia photoreceptorwere absent in both connecting ift88 and cilia kif3a were mutants (Figure 4A). These results further confirmed that photoreceptor connecting cilia initially formed but failed to maintain in ift74 mutants, which is different from severe were initially formed but failed to maintain in ift74 mutants, which is different from severe ciliogenesisciliogenesis defects defects in inift88 ift88 and kif3akif3a mutants.mutants.

Figure 4. Photoreceptor connecting cilia in ift74, ift88, and kﬁ3a mutants. (A) Confocal images showing photoreceptor connecting cilia in the retinae of wild-type and mutant larvae at different stages as indicated. Cilia were stained with anti-acetylated alpha tubulin antibody (green). Arrows indicate the connecting cilia. (B,C) Dot plots showing the number

and length of connecting cilia in different groups as indicated. Scale bar: 10 µm. *** p < 0.001, **** p < 0.0001, ns, no signiﬁcant. Int. J. Mol. Sci. 2021, 22, 9329 7 of 14

Figure 4. Photoreceptor connecting cilia in ift74, ift88, and kfi3a mutants. (A) Confocal images showing photoreceptor Int. J. Mol. Sci. 2021 22 connecting, , 9329cilia in the retinae of wild‐type and mutant larvae at different stages as indicated. Cilia were stained with anti‐ 7 of 13 acetylated alpha tubulin antibody (green). Arrows indicate the connecting cilia. (B,C) Dot plots showing the number and length of connecting cilia in different groups as indicated. Scale bar: 10 μm. *** p < 0.001, **** p < 0.0001, ns, no significant.

2.5.2.5. Defects Defects of of Opsin Opsin TransportTransport Machinery Machinery in in ift74 ift74 Mutant Mutant Retina Retina OpsinOpsin mislocalization mislocalization is is one one of the of main the main causes causes of photoreceptor of photoreceptor cell death. cellTo deter death.‐ To determinemine the reasons the reasons for photoreceptor for photoreceptor degeneration degeneration in the ift74 in mutant the ift74 retinae,mutant we further retinae, we furtheranalyzed analyzed the transport the transport efficiency efficiency of opsin. of We opsin. performed We performed a pulse‐chase a pulse-chase experiment experiment by byinjecting injecting zebrafish zebrafish embryos embryos with with hsp:GFPhsp:GFP-CT44‐CT44 construct,construct, which which encodes encodes a GFP a protein GFP protein fusedfused with with the the 44 44 C-terminal C‐terminal amino amino acids acids of of xenopus xenopus opsin driven by by a a heat heat shock shock pro promoter.‐ moter. In photoreceptor cells, the GFP‐CT44 chimeric protein is transported to the outer In photoreceptor cells, the GFP-CT44 chimeric protein is transported to the outer segment, segment, which is similar to the localization of endogenous rhodopsin [26]. We injected which is similar to the localization of endogenous rhodopsin [26]. We injected this construct this construct into one‐cell stage zebrafish embryos collected from two intercrossed ift74 into one-cell stage zebrafish embryos collected from two intercrossed ift74 heterozygotes. heterozygotes. At 5 dpf, the injected embryos were heat induced for one◦ hour at 37 °C. In Atcontrol 5 dpf, thesiblings, injected the embryosmajority of were GFP heat signals induced were cleared for one from hour the at 37innerC. segments In control and siblings, theaccumulated majority of in GFP the outer signals segments were clearedas early fromas 4 h theafter inner heat inducement segments and (Figure accumulated 5A). In in thecontrast, outer segments a large amount as early of GFP as 4 signals h after were heat still inducement present in the (Figure inner5 segmentsA). In contrast, of photo a‐ large amountreceptor of cells GFP of signals the ift74 were mutants still present at 4 h after in the induction inner segments(Figure 5B). of Interestingly, photoreceptor the cells chi‐ of the ift74mericmutants protein at became 4 h after concentrated induction in (Figure the outer5B). segments Interestingly, at 24 h the after chimeric heat shock protein in both became concentratedmutants and in control the outer siblings segments (Figure 5C–E). at 24 h after heat shock in both mutants and control siblings (Figure5C–E).

Figure 5. Opsin transport analysis in ift74 mutant. (A–D) Representative confocal images of transverse cryosections through Figure 5. Opsin transport analysis in ift74 mutant. (A–D) Representative confocal images of transverse cryosections the centralthrough retinae the central of wild-type retinae of and wildift74‐typemutant and ift74 larvae mutant at 4 larvae and 24 at h4 and after 24 heat-shock h after heat induction.‐shock induction. The subcellular The subcellular distribution of thedistribution GFP-CT44 of (green) the GFP was‐CT44 evaluated. (green) was Sections evaluated. were Sections counterstained were counterstained with phalloidin with phalloidin (red) to visualize (red) to visualize the outer the limiting membraneouter andlimiting the outermembrane plexiform and the layer. outer Cell plexiform nuclei were layer. stained Cell nuclei with were DAPI stained in blue. with (E )DAPI Relative in blue. GFP ( fluorescenceE) Relative GFP intensity in thefluorescence cell bodies measuredintensity in betweenthe cell bodies the outer measured limiting between membrane the outer and limiting the outer membrane plexiform and layer.the outer Asterisks plexiform indicate layer. outer Asterisks indicate outer segments; arrowheads indicate the outer limiting membrane and arrows indicate the outer plexi‐ segments;form layer. arrowheads Scale bar: indicate 10 μm. **** the p outer < 0.0001, limiting ns, no significant. membrane and arrows indicate the outer plexiform layer. Scale bar: 10 µm. **** p < 0.0001, ns, no significant. Opsin mislocalization in the mutant photoreceptor cells may due to the overex‐ pressedOpsin fusion mislocalization proteins in insome the photoreceptors mutant photoreceptor containing cells an excess may due amount to the of overexpressed injected fusionconstruct. proteins To exclude in some such photoreceptors possibility, we containing further generated an excess a amount stable line of injectedTg(hsp:GFP construct.‐ To exclude such possibility, we further generated a stable line Tg(hsp:GFP-CT44) and crossed it with a transgenic line Tg(sws1:HA-tdTomato-CT44). The Tg(sws1:HA-tdTomato- CT44) transgenic line contains a similar opsin localization signal fused to the C-terminal of tdTomato, which is driven by the promoter of the UV-sensitive opsin gene sws1 [11,27]. We injected ift74 morpholinos into the embryos carrying both transgenes. When measuring the GFP or tdTomato signals’ intensity, we found an ectopic localization of these chimeric proteins in the mutant inner segments at both 4 h and 24 h after heat shock (Figure S5). Taken together, these results suggest that opsin transport efficiency was compromised in both ift74 mutants and morphants despite the presence of cilia at early stages. Int. J. Mol. Sci. 2021, 22, 9329 8 of 13

2.6. Maternal Ift74 Proteins Contribute to the Early Development of Photoreceptors In ift74 mutants, ciliogenesis defects occurred as early as 3 dpf in multiple tissues, including olfactory epithelium, spinal cord, and pronephric duct (Figure S2). Considering that Ift74 is an essential member of the IFT-B core complex, it is quite unexpected that the connecting cilium initially developed in the mutants, only leading to slow progressing degeneration. We think it is possible that maternal Ift74 proteins may contribute to the initial development of photoreceptors. To test this hypothesis, we evaluated the expression of maternal ift-b genes (encoding members of the IFT-B complex) by real-time quantitative PCR (RT-qPCR) analysis. By using total RNA extracted from single cell stage embryos, we showed that ift52, ift57, ift74, ift81, ift88, and ift172 were all expressed maternally. The expression level of maternal deposited ift74 gene was signiﬁcantly higher than those of other ift-b genes (Figure6A). To prevent the expression of maternal genes, we used ift74 morpholinos (ATG-MO) to block the translation of both maternal and zygotic ift74 genes by targeting the translational start region. In wild-type larvae, knockdown of the ift74 gene with ATG- and SP-morpholinos inhibited the development of connecting cilium, which is much more severe than those of ift74 mutants (Figure6B–D). Then, we injected ATG-MO into ift74 mutants to block the translation of maternal ift74 transcripts, which exacerbated the severity of connecting cilium development (Figure6B–D). Furthermore, photoreceptor degeneration was also enhanced in the absence of maternal transcripts in ift74 mutants (Figure6E,F). Finally, opsin proteins were still present in the inner segments at 24 h after heat inducement in ift74 morphants, which is different to those of ift74 mutants (Figure5 Int. J. Mol. Sci. 2021, 22, 9329 9 of 14 and Figure S5). Together, these results suggested that maternal deposit ift74 genes may be the main reason of the slow progressing photoreceptor degeneration in the mutants.

FigureFigure 6. Maternal 6. Maternal Ift74 Ift74 contributes contributes to the the slow slow photoreceptor photoreceptor degeneration degeneration.. (A) qRT‐ (PCRA) qRT-PCR results showing results relative showing expres relative‐ expressionsion level level of of iftift-b‐b genesgenes in one in one-cell‐cell stage stage embryos. embryos. The expression The expression of the ift74 of gene the ift74 was geneset as was100%. set (B as) Confocal 100%. (imagesB) Confocal imagesshowing showing photoreceptor photoreceptor connecting connecting cilia in cilia the in retinae the retinae of 5 dpf of wild 5 dpf‐type, wild-type, ift74 morphants,ift74 morphants, and mutant and larvae. mutant Cilia larvae. were Cilia stained with anti‐acetylated alpha tubulin antibody (green). (C,D) Dot plots showing the number and length of connecting were stained with anti-acetylated alpha tubulin antibody (green). (C,D) Dot plots showing the number and length of cilia in different groups as indicated. (E) Confocal images showing rod outer segments visualized with zpr‐3 antibody in connecting5 dpf ift74 cilia mutants in different injected groups with control as indicated. or ATG ( morpholinoE) Confocal as images indicated. showing Enlarged rod views outer of segmentsthe boxed area visualized are shown with on zpr-3 antibodythe inright. 5 dpf (F)ift74 Statisticalmutants results injected showing with the control number or of ATG outer morpholino segments per as retina indicated. section Enlarged in different views groups of the as indicated. boxed area are shownScale on the bars: right. B, 5 μ (Fm;) StatisticalE, 30 μm. * resultsp < 0.05, showing ** p < 0.01, the *** numberp < 0.001, of**** outer p < 0.0001, segments ns, no per significant. retina section in different groups as indicated. Scale bars: B, 5 µm; E, 30 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no signiﬁcant. 3. Discussion Being a key member of the core IFT‐B complex, the role of Ift74 during intraflagellar transport has been extensively investigated, while less is known about its roles during early embryonic development. In this study, we reported the role of Ift74 protein during early zebrafish development. Mutation of the ift74 gene led to body curvature defects, which is a characteristic phenotype of ciliary mutants. The formation of a curly body axis was caused by abnormal urotensin signals originated from cilia‐driven cerebrospinal fluid flow [23,28]. In line with this, we found ciliogenesis defects in multiple organs in the mu‐ tants as early as 3 dpf (Figure S2). Noticeably, the severity of ciliary defects was different among these organs. Nearly all the cilia were absent in the spinal canal and olfactory epi‐ thelium, while cilia were maintained but shorter in ear hair cells and pronephric ducts, suggesting different requirement of Ift74 proteins in these tissues during cilia formation. Unexpectedly, ift74 mutants displayed slow progressing photoreceptor degenera‐ tion. Both rod and cone photoreceptors were grossly normal in the mutants at 3 and 5 dpf, but they gradually degenerated with development. Some photoreceptor cells can survive as late as 10 dpf in the mutant retinae. These phenotypes were very different from the rapid photoreceptor degeneration in ift88 and kif3a mutants (Figure S4). In other IFT‐B mutants (ift57, ift70 and ift172), rapid photoreceptor degeneration also occurred at early stages [14–17]. To the best of our knowledge, this is the first IFT‐B mutant that displays slow photoreceptor degeneration in zebrafish. Actually, the photoreceptor defects were similar to those observed in ift122 mutants, an IFT‐A component, while no body curvature

Int. J. Mol. Sci. 2021, 22, 9329 9 of 13

3. Discussion Being a key member of the core IFT-B complex, the role of Ift74 during intraflagellar transport has been extensively investigated, while less is known about its roles during early embryonic development. In this study, we reported the role of Ift74 protein during early zebrafish development. Mutation of the ift74 gene led to body curvature defects, which is a characteristic phenotype of ciliary mutants. The formation of a curly body axis was caused by abnormal urotensin signals originated from cilia-driven cerebrospinal fluid flow [23,28]. In line with this, we found ciliogenesis defects in multiple organs in the mutants as early as 3 dpf (Figure S2). Noticeably, the severity of ciliary defects was different among these organs. Nearly all the cilia were absent in the spinal canal and olfactory epithelium, while cilia were maintained but shorter in ear hair cells and pronephric ducts, suggesting different requirement of Ift74 proteins in these tissues during cilia formation. Unexpectedly, ift74 mutants displayed slow progressing photoreceptor degeneration. Both rod and cone photoreceptors were grossly normal in the mutants at 3 and 5 dpf, but they gradually degenerated with development. Some photoreceptor cells can survive as late as 10 dpf in the mutant retinae. These phenotypes were very different from the rapid photoreceptor degeneration in ift88 and kif3a mutants (Figure S4). In other IFT-B mutants (ift57, ift70 and ift172), rapid photoreceptor degeneration also occurred at early stages [14–17]. To the best of our knowledge, this is the first IFT-B mutant that displays slow photoreceptor degeneration in zebrafish. Actually, the photoreceptor defects were similar to those observed in ift122 mutants, an IFT-A component, while no body curvature defects were reported in ift122 mutants, implying the different mechanisms in ciliogenesis defects between IFT-A and IFT-B mutants [18]. The survival of photoreceptors relies on the presence of photoreceptor connecting cilium, through which opsin proteins can be transported to the outer segment. Consistent to the slow photoreceptor degeneration, connecting cilium was also formed at early stages, while it failed to maintain in the mutants. Opsin mislocalization was one of the major causes for photoreceptor cell death. We previously showed that the failure of connecting cilium development led to an ectopic localization of rhodopsin in the inner segments, where it activated intracellular calcium signals and finally resulted in cell death [11]. The presence of connecting cilium ensured the transport of opsin proteins in the mutant retinae and thus prevented the degeneration of photoreceptors at early stages. On the other hand, transient and stable transgenic analysis using GFP-tagged opsin proteins showed that opsin transport efficiency was still compromised in the mutants. In the retinae of wild-type larvae, opsin proteins are transported quickly as the majority of opsin proteins started to accumulate in the outer segments as early as 4 h after heat inducement. In contrast, opsin remained in the inner segment of mutant photoreceptors at 4 h after inducement. Interestingly, opsin transport was still functional, as the majority of opsin can be transported to the outer segments at 24 h after heat inducement in the mutant retinae. Together, these data suggested that opsin transport was defective with lower transport efficiency in the ift74 mutants, which may represent the major reason accounting for slow photoreceptor degeneration. In the ift74 mutants, ciliogenesis defects occurred in multiple organs as early as in 3 dpf zebrafish larvae. It is surprising that photoreceptor connecting cilia were formed and maintained at 5 dpf. Even at 7 dpf, the remaining connecting cilia were still of similar length to those of wild-type larvae. It is clear that the formation and maintenance of connecting cilia depend on Ift74 proteins, considering opsin transport defects in the mutant. Interestingly, we found that the expression level of maternal ift74 transcripts was significantly higher than those of other ift-b genes in one-cell stage zebrafish embryos. Maternal message RNAs play essential roles during maternal to zygotic transition to ensure the normal development of zebrafish embryos. These maternal ift mRNAs ensure the proper development of cilia during early stages. For instance, cilia were present in zygotic ift88 (oval) mutants at 24 h post fertilization, while all cilia were absent in the MZift88 mutants lacking both maternal and zygotic Ift88 proteins [29]. The presence of photoreceptor connecting cilia Int. J. Mol. Sci. 2021, 22, 9329 10 of 13

may due to the excess deposit of ift74 maternal transcripts compared with those of other ift-b genes. Further knocking down of maternal ift74 genes with ift74 ATG-MO exacerbated photoreceptor degeneration in the mutants, which confirmed the importance of maternal transcripts. Interestingly, only the connecting cilia can be maintained for a while in the mutants, while all other types of cilia were still defective at earlier stages. It is possible that the photoreceptor connecting cilia were shorter and stable to ensure the fast transport of opsin proteins, where only a small amount of maternal proteins were sufficient to perform such roles. While in other tissues, the maintenance of entire cilia requires many more Ift74 proteins. The lower opsin transport efficiency in the mutants further confirmed the minimum requirement of Ift74 proteins for photoreceptor survival. Moreover, it is possible that the Ift74 degradation rate was slower in the photoreceptor cells, leading to the presence of a large amount of Ift74 proteins than other tissues. In humans, IFT74 deficiency has been implicated in several ciliopathies, including BBS and JBTS [20–22]. Recently, male sterility due to impaired flagellogenesis was also reported [30]. In contrast to the gross ciliary defects in zebrafish ift74 mutants, the clinical manifestations of human patients with IFT74 mutations were quite variable. BBS patients are characterized by retinal dystrophy, obesity, polydactyly, and renal dysfunction, while JBTS patients have a distinctive cerebellar and brain stem malformation called the molar tooth sign (MTS) [31,32]. Interestingly, both JBTS and BBS IFT74 patients displayed retinal defects, suggesting an essential function of this protein during retinal development. The pleiotropicity of these ciliopathies may be caused by the variety of IFT74 mutations. For instance, we found that the BBS variant of IFT74 (∆561–600-IFT74) lost its binding ability with IFT27, which is a Small GTPase-like protein required for BBSome function [33,34], while the JBTS variant (p. Q179E) maintains its binding activity with IFT27 [20]. It is apparent that the IFT74 variants reported in humans are hypomorphic, while our zebrafish mutants are likely to be a null allele, causing ciliogenesis defects in almost every organ. In the future, it is worth generating zebrafish mutants harboring different ift74 variants or transgenic lines expressing these human variants in ift74 homozygous mutants, which may provide further answers for the subtle difference of these IFT74 variants in different organs. In summary, we showed that Ift74 was necessary for ciliogenesis in zebrafish. Malfunc- tion of Ift74 resulted in ciliogenesis defects in the majority of organs, while photoreceptor connecting cilia were initially formed and degenerated slowly in the mutants. Compared with other genes encoding IFT-B complex, zebrafish eggs deposit more maternal ift74 transcripts, representing the main causes of slow photoreceptor degeneration of the mutants.

4. Materials and Methods 4.1. Zebrafish Strain All experiments involving zebrafish strains were maintained at 28 ◦C on a 14 h light/10 h dark cycle. Embryos were raised at 28.5 ◦C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.39 mM CaCl2, 0.67 mM MgSO4, 0.1% methylene blue (Sinopharm, Qingdao, China)) following standard protocols. CRISPR/Cas9 technology was used to generate zebrafish ift74 mutants with the following target site for single guide (sg) RNA (50-GTGCCCGGACGGGCCGTCCC-30). The sgRNA was designed using ZiFit targeter (http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx) (accessed on 5 December 2017). The Cas9 mRNA and sgRNA synthesis were similar to previously described [28,35]. Guide sgRNAs and Cas9 mRNA were co-injected into zebrafish embryos at one-cell stage.

4.2. Genetic Mapping and Cloning A map cross was set up between heterozygous carriers of michelin allele (Tübingen genetic background) and wild-type IND strain homozygotes. The michelin locus was mapped in a F2 intercross using bulked segregant analysis. DNA samples were genotyped by PCR with SSLP markers evenly spaced across zebraﬁsh genome. To determine the michelin locus, we used a panel of 24 F2 diploid embryos obtained via incrossing of F1 adult animals. To determine the segregation patter of genomic polymorphisms, DNA samples Int. J. Mol. Sci. 2021, 22, 9329 11 of 13

were genotyped by PCR with SSLP markers evenly spaced across the F2 embryos’ genome. PCR products were electrophoresed on 4% agarose gels.

4.3. Whole-Mount In Situ Hybridization and Immunohistochemistry For in situ hybridization, ift74 gene was cloned from a wild-type cDNA library into pGEM-T vector, linearized with a restriction enzyme, and then transcribed using SP6 poly- merase and digoxigenin-labeled UTP (Roche, Mannheim, Germany). Probe preparation and hybridization were carried out using standard protocols. For immunohistochemistry, zebrafish larvae were fixed in 4% paraformaldehyde (Sigma-Aldrich, Saint Louis, MO, USA) in phosphate-buffered saline (PBS, Sangon Biotech, Shanghai, China) overnight at 4 ◦C. Fixed larvae were cryoprotected using 30% sucrose in PBS, embedded in OCT (Leica, Nussloch, Germany), frozen, and sectioned at 12 µm thickness. Sections through the central retinae were placed on slides, dried for 2 h at 37 ◦C, rehydrated in PBST for 5 min, incubated in blocking solution (5% normal goat serum (G-clone, Beijing, China) and 0.5%Txiton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) in PBST) for 30 min at room temperature, and incubated with primary antibodies in blocking solution overnight at 4 ◦C. Slides were washed with PBS three times for 10 min each and incubated with second antibodies for 2 h at room temperature. Antigen retrieval was performed on connecting cilia staining by incubating with 10 mM sodium citrate for 30 min at 65 ◦C, which was followed by regular immunostaining protocol. The following antibodies were used: anti-acetylated α-tubulin (1:500, Cell Signaling Technology, Danvers, MA, USA, #5335S), anti-glycylated tubulin (1:500, Millipore, Burlington, MA, USA), zpr-3 (1:500, Zebrafish International Resource Center, Eugene, OR, USA), zpr-1 (1:500, Zebrafish International Resource Center, Eugene, OR, USA). After staining, the confocal images were collected on a Leica TCS Sp8 confocal microscope.

4.4. Quantitative PCR The primers used for qPCR are summarized in Supplemental Table S1. The PCR reaction was set up using Eva-Green Master Mix (ABM, Richmond, BC, Canada) and performed on a Step One real-time PCR system (Thermo Scientific, Waltham, MA, USA). The amplification condition parameters were 95 ◦C for 15 s, followed by 40 cycles of 95 ◦C for 5 s, 60 ◦C for 15 s, and 72 ◦C for 35 s, and each sample had a triplicate reaction. Relative gene express levels were quantified using the comparative Ct method (2−∆∆Ct method) based on Ct values for target gene and zebrafish β-actin.

4.5. Morpholino Knockdown To knockdown the expression of the ift74 gene, we used a morpholino targeting to the 50 untranslated region (ATG-MO) and an anti-splice site morpholino (SP-MO). These morpholinos were the same as described previously [20] (Table S1).

4.6. Opsin Transport Assay The hsp:GFP-CT44 chimeric construct was injected into ift74 mutants or sibling embryos at the one-cell stage. For stable line analysis, we microinjected ATG and SP MOs into one-cell stage embryos collected from crossing between the Tg(hsp:GFP-CT44) and Tg(sws1:HA-tdTomato-CT44) transgenic line. The injected larvae were heat-shocked at 5 dpf for 1 h at 37 ◦C; then, they were ﬁxed at 4 and 24 h after heat shock and cryosec- tioned with standard protocol. Sections were counter-stained with Alexa Fluor™ 546 Phalloidin(Invitrogen, Waltham, MA, USA) and imaged using a confocal microscope (SP8, Lecia, Wetzlar, Germany). Fluorescence signals were measured using ImageJ software(version1.53c, National Institutes of Health, Bethesda, MD, USA), and signals in the cell bodies were measured between the outer limiting membrane and the outer plexiform layer. The relative signal intensity of the cell body in each photoreceptor cell was calculated as percentages of ﬂuorescence signal intensity between the cell body and entire cell. Int. J. Mol. Sci. 2021, 22, 9329 12 of 13

4.7. Statistical Analysis All experiments were repeated at least three times. Scatterplot data are presented as mean ± S.D. as indicated in the figure legends. Statistical significance was evaluated using Student’s t-test, and p < 0.05 was taken to be statistically significant. All statistical analysis was performed using GraphPad Prism 8 software(version 8.0.2, San Diego, CA, USA).

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22179329/s1. Figure S1: PCR analysis of ift74 locus in michelin mutants, Figure S2: Cilia defects in 3dpf ift74 mutants, Figure S3: Phenotypes of photoreceptor outer segments in ift74 mutants, Figure S4: Rapid photoreceptor degeneration in ift88 and kif3a mutants, Figure S5: Opsin transport defects in ift74 morphants, Table S1: List of primers used in this study. Author Contributions: Conceptualization, P.Z., J.X. and C.Z.; methodology, P.Z. and C.Z.; software, P.Z.; validation, J.X. and C.Z.; formal analysis, P.Z., J.X. and C.Z.; investigation, P.Z. and J.X.; resources, Y.W. and P.Z.; data curation, P.Z., J.X., Y.W. and C.Z.; writing—original draft preparation, P.Z. and C.Z.; writing—review and editing, J.X., Y.W. and C.Z.; visualization, P.Z.; supervision, C.Z.; project administration, C.Z.; funding acquisition, C.Z. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Fundamental Research Funds for Central Universi- ties of China (Grants 202064009, 201941004, and 201961016) and the grant from Laboratory for Marine Biology and Biotechnology of Qingdao National Laboratory for Marine Science and Technol- ogy (MS2019NO02). Institutional Review Board Statement: All zebrafish study was conducted according standard animal guidelines and approved by the Animal Care Committee of Ocean University of China (Animal protocol number: OUC2012316). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in article and Supplementary Material. Acknowledgments: The authors thank Shunji Jia and Hongbin Jin for their help during the initial mapping of the michelin mutants and members of the Zhao’s lab for their excellent supports during the preparation of this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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